【摘要】：食源性致病菌易引起食源性疾病,是影響食品安全的主要因素之一。但傳統(tǒng)的食源性致病菌的鑒定和檢測方法在一定程度上存在檢測步驟繁瑣、耗時費力、穩(wěn)定性差等不足,難以滿足食品安全快速、準確的檢測要求。時間分辨熒光技術可根據(jù)熒光物質(zhì)壽命的長短,消除背景熒光,進而提高檢測信噪比和靈敏度。目前在臨床醫(yī)學領域已得到廣泛的應用,然而在食品安全檢測領域的應用還比較少,本研究采用溶劑熱法制備了兩種具有時間分辨熒光特性的納米材料,結合適配體技術、磁分離技術和熒光共振能量轉移技術,構建了一系列快速、準確、靈敏的食源性致病菌檢測新方法。首先,采用溶劑熱法合成了具有時間分辨熒光特性的Na YF_4:Ce/Tb納米材料(TRNPs),結合鼠傷寒沙門氏菌適配體識別元件和時間分辨熒光共振能量轉移(TR-FRET)技術,構建了基于時間分辨熒光納米材料和熒光染料共振能量轉移檢測鼠傷寒沙門氏菌的方法。在最優(yōu)實驗條件下,檢測線性范圍為10~2~10~6 cfu/m L(y=0.796-0.096x,R~2=0.9932),檢測限為25 cfu/m L。將本方法應用于實際樣品中,通過特異性研究和與平板計數(shù)方法的比較,表明該方法可用于實際樣品的檢測。其次,合成了表面氨基化的Fe_3O_4磁性納米材料(MNPs)和另一種具有時間分辨熒光特性的Na Gd F_4:Eu納米材料(TRNPs)。Na Gd F_4:Eu納米材料與Na YF_4:Ce/Tb納米材料可用同一波長的激發(fā)光進行激發(fā),發(fā)射光譜不重疊。通過將鼠傷寒沙門氏菌和金黃色葡萄球菌適配體分別修飾兩種TRNPs和MNPs構成熒光探針和捕獲探針,構建了基于雙色時間分辨熒光納米材料熒光標記和磁分離技術同時檢測鼠傷寒沙門氏菌和金黃色葡萄球菌的方法。在最佳實驗條件下,鼠傷寒沙門氏菌檢測線性范圍為10~2~10~5cfu/m L(y=10213x-12208.92,R~2=0.9922),檢測限為15 cfu/m L,金黃色葡萄球菌檢測線性范圍為10~2~10~5 cfu/m L(y=4803.20x-1933.87,R~2=0.9982),檢測限為20 cfu/m L。將本方法應用于實際樣品中,結果與平板計數(shù)法一致性良好,驗證了本方法的實際應用性。最后,利用合成的Na YF_4:Ce/Tb和Na Gd F_4:Eu納米材料,結合二硫化鎢(WS_2)的高效熒光淬滅性能,建立了基于雙色時間分辨熒光納米材料和WS_2熒光共振能量轉移同時檢測鼠傷寒沙門氏菌和金黃色葡萄球菌的方法。在最優(yōu)實驗條件下,鼠傷寒沙門氏菌檢測線性范圍為10~2~10~6 cfu/m L(y=8670.59x-11372.41,R~2=0.9905),檢測限為20cfu/m L;金黃色葡萄球菌檢測線性范圍為10~2~10~(6 )cfu/m L(y=4613.55x-5332.39,R~2=0.9962),檢測限為33 cfu/m L。將本方法應用于實際樣品的檢測,結果與平板計數(shù)法一致性良好,驗證了本方法的實際應用性。
[Abstract]:Food-borne pathogens are easy to cause food-borne diseases, which is one of the main factors affecting food safety. However, the traditional methods of identification and detection of foodborne pathogenic bacteria have some shortcomings, such as tedious detection steps, time-consuming and laborious, poor stability and so on, so it is difficult to meet the requirements of food safety, rapid and accurate detection. Time-resolved fluorescence technique can eliminate background fluorescence and improve detection SNR and sensitivity according to the lifetime of fluorescence material. At present, it has been widely used in the field of clinical medicine, but it is less used in the field of food safety detection. In this study, two kinds of nanomaterials with time-resolved fluorescence characteristics were prepared by solvothermal method, combined with aptamer technology. A series of rapid, accurate and sensitive methods for the detection of foodborne pathogenic bacteria were constructed by magnetic separation and fluorescence resonance energy transfer. Firstly, the Na YF_4:Ce/Tb nanomaterials (TRNPs), with time-resolved fluorescence properties were synthesized by solvothermal method, combined with an aptamer recognition element of Salmonella typhimurium and a time-resolved fluorescence resonance energy transfer (TR-FRET) technique. A method for detecting Salmonella typhimurium based on time resolved fluorescent nanomaterials and fluorescence dye resonance energy transfer was developed. Under the optimum experimental conditions, the linear range of detection was 10 ~ 2 ~ 10 ~ (10) cfu/m / L (~ (0.796-0.096) x ~ (+) ~ (2) ~ (0.9932) and the detection limit was 25 cfu/m 路L ~ (-1). The method has been applied to practical samples, and compared with the plate counting method, it is proved that this method can be used for the detection of real samples. Secondly, The surface amino Fe_3O_4 magnetic nanomaterials (MNPs) and another Na Gd F_4:Eu nanomaterial (TRNPs). Na Gd F_4:Eu nanomaterials with time-resolved fluorescence properties and Na YF_4: were synthesized. Ce/Tb nanomaterials can be excited by excitation light of the same wavelength. The emission spectrum does not overlap. Two kinds of TRNPs and MNPs were modified with Salmonella typhimurium and Staphylococcus aureus aptamer respectively to form fluorescence probe and capture probe. A method for simultaneous detection of Salmonella typhimurium and Staphylococcus aureus was developed based on fluorescence labeling and magnetic separation of two color time-resolved fluorescent nanomaterials. Under the optimum experimental conditions, the linear range of detection of Salmonella typhimurium was 10~2~10~5cfu/m L (yttrix 10213x-12208.92) 0.9922, the detection limit was 15 cfu/m / L, and the linear range of detection of Staphylococcus aureus was 100.803.20x-1933.87 cfu/m / L. The detection limit was 20 cfu/m L. The method is applied to practical samples, and the result is in good agreement with the plate counting method, which verifies the practical application of this method. Finally, the high performance fluorescence quenching properties of tungsten disulfide (WS_2) were studied by using the synthesized Na YF_4:Ce/Tb and Na Gd F_4:Eu nanomaterials. A method for simultaneous detection of Salmonella typhimurium and Staphylococcus aureus based on two-color time-resolved fluorescent nanomaterials and WS_2 fluorescence resonance energy transfer was developed. Under the optimum experimental conditions, the linear range of detection of Salmonella typhimurium was 100.59x-11372.41 cfu/m / L (YY 8670.59x-11372.41), and the detection limit was 20cfu/m L. The linear range of detection of Staphylococcus aureus was 10 ~ (2) ~ (6) cfu/m / L (YP4613.55x-5332.39) and the detection limit was 33 cfu/m / L. The method is applied to the detection of practical samples, and the results are in good agreement with the plate counting method, which verifies the practical application of this method.
【學位授予單位】：江南大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：O657.3;TS207.4

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